This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2. IRPs bind IRE's when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and other IRE containing transcripts and prolongation of the half-life of the transferrin receptor mRNA. IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. Regulation of RNA binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound, to a form that loses both iron and aconitase activity. The [4Fe-4S] containing protein does not bind IREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to determine whether IRP1 will bind RNA. Recently, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS and Nif U genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron-sulfur cluster of IRP1. IRP2 also binds IREs in iron-depleted cells, but unlike IRP1, IRP2 is degraded in cells that are iron-replete. Experimental evidence indicates that IRP2 binds iron and undergoes iron-catalyzed oxidation. The oxidized protein is then selectively ubiquitinated and degraded by the proteasome. Indirect evidence suggests that numerous other proteins will be degraded by a pathway in which oxidative modification is followed by ubiquitination and proteasomal degradation of the ubiquitinated substrate. To approach questions about the physiology of iron metabolism, loss of function mutations of IRP1 and IRP2 have been generated in mice through homologous recombination in embryonic cell lines. In the absence of provocative stimuli, there are no abnormalities in iron metabolism associated with loss of IRP1 function. IRP2-/- mice develop a progressive neurologic syndrome characterized by gait abnormalities and tremor. Loss of function of both IRPs appears to be lethal. Characterization of the roles of the genes for hereditary hemochromatosis and murine microcytic anemia are ongoing in normal and knockout mice.